Regulation of synapse density and E/I balance by NFAT-dependent transcription

项目总结/摘要 突触的形成和消除不仅对电路的发展至关重要，对经验也至关重要- 依赖性回路重塑和维持兴奋/抑制（E/I）平衡。这些调节失调 过程，不成比例的突触密度，和E/I失衡是常见的多个神经系统 包括自闭症谱系障碍（ASD）、阿尔茨海默氏病（AD）和精神分裂症在内的疾病 （SCZ）。然而，我们对调节突触密度和E/I比的机制的理解是不完整的。 兴奋性和抑制性突触都受到活动依赖性突触可塑性的影响;模式化活动 可以诱导突触形成或消除，或者可以产生结构、分子 组成和突触强度。重要的是，它们不是孤立地发挥作用，而是， 它们相互协作以维持神经元的功能，并且其中一个的变化常常伴随着另一个的变化。 活动依赖性突触改变是由快速的翻译后修饰和亚细胞介导的。 重新分配现有蛋白质。这种变化是由蛋白质合成长期支持的， 需要从头mRNA合成。电活动和突触后Ca 2+信号传导与转录的耦合 被称为兴奋-转录（E-T）偶联：许多形式的L-型下游作用 电压门控钙通道（LTCCs）。一种形式的E-T耦合已经被我们的研究人员很好地表征， 通过活化T细胞核因子（NFAT）家族转录因子的活化来产生实验室信号。 LTCC Ca 2+内流激活Ca 2 +/CaM依赖性蛋白磷酸酶2B/钙调磷酸酶 (PP2B/CaN），其通过A-激酶锚定蛋白79/150（AKAP 79/150）定位于通道，并且反过来 CaN使NFAT去磷酸化以促进其核转位。有证据表明，这种失调 通路可能参与突触密度的病理性改变，这一假设得到了最近的研究的支持。 我们实验室的初步数据然而，NFAT信号是否调节突触密度和E/I平衡， 在神经系统疾病中这一点如何改变仍不清楚。 因此，我建议测试CaN-NFAT信号调节突触密度和E/I的假设， Aβ可以激活这一通路，改变AD中的这些突触特性。 临床损害几乎完全源于病理性突触消除。我将主要使用 荧光显微镜和电生理学来确定增强的CaN-NFAT信号传导对 兴奋性和抑制性突触密度和E/I突触比（目的1），并确定Aβ是否诱导CaN- NFAT信号传导和NFAT依赖性转录以改变突触密度（Aim 2）。

PROJECT SUMMARY / ABSTRACT Synapse formation and elimination are critical not only to circuit development, but also to experience- dependent circuit remodeling and to maintenance of excitatory/inhibitory (E/I) balance. Dysregulation of these processes, disproportionate synapse density, and E/I imbalance are common to multiple nervous system disorders including Autism Spectrum Disorders (ASDs), Alzheimer’s Disease (AD) and Schizophrenia (SCZ). However, our understanding of the mechanisms regulating synapse density and E/I ratio is incomplete. Both excitatory and inhibitory synapses are subject to activity-dependent synaptic plasticity; patterned activity can induce synapse formation or elimination, or can produce persistent changes in structure, molecular composition, and synaptic strength. Importantly, they do not function in isolation of one another, but rather, they cooperate to maintain neuronal function and changes at one are often accompanied by changes at the other. Activity-dependent synaptic alterations are initiated by rapid posttranslational modification and subcellular redistribution of existing proteins. Such changes are supported long-term by protein synthesis, which often requires de novo mRNA synthesis. Coupling electrical activity and postsynaptic Ca2+ signaling to transcription is known as excitation-transcription (E-T) coupling: many forms of which operate downstream of L-type voltage-gated Ca2+ channels (LTCCs). One form of E-T coupling that has been well characterized by our laboratory signals via activation of the nuclear factor of activated T-cells (NFAT) family of transcription factors. LTCC Ca2+ influx activates Ca2+/calmodulin (Ca2+/CaM)-dependent protein phosphatase 2B/calcineurin (PP2B/CaN) that is localized to the channel by A-kinase-anchoring protein 79/150 (AKAP79/150), and in turn CaN dephosphorylates NFAT to promote its nuclear translocation. Evidence suggests that dysregulation of this pathway may be involved in pathological alterations to synapse density—a hypothesis that is supported by recent preliminary data from our lab. However, whether NFAT signaling regulates synapse density and E/I balance, and how this may be altered in nervous system disorders remain unclear. Thus, I propose to test the hypotheses that CaN-NFAT signaling regulates synapse density and E/I ratio and that Aβ can activate this pathway to alter these synaptic properties in AD—a disorder whose clinical impairment stems almost entirely from pathological synapse elimination. I will primarily use fluorescence microscopy and electrophysiology to determine the effect of enhanced CaN-NFAT signaling on excitatory and inhibitory synapse density and E/I synaptic ratio (Aim 1), and to determine if Aβ induces CaN- NFAT signaling and NFAT-dependent transcription to alter synapse density (Aim 2).